The broad, long-term objective of the research proposed in this application is to understand the cellular and molecular mechanisms that determine how synapses work. One of the most complex events occurring in a synapse is the synaptic vesicle cycle. Some aspects of the cycle are understood in molecular detail;the least understood aspect concerns the mobilization of synaptic vesicles, that is, their movement from deep in the cytoplasm to the presynaptic membrane as a prelude to docking and exocytosis. The Specific Aims of this proposal are to monitor the movements of synaptic vesicles in living synapses, and identify the molecular mechanisms that regulate.the motion. .. ... We will directly monitor synaptic vesicle movements in motor nerve terminals of the neuromuscular junction (NMJ). We will selectively label distinct vesicle pools in living nerve terminals with fluorescent (FM) dyes, and monitor vesicle movements at rest, during nerve stimulation, and after applying drugs that interfere with specific signaling pathways. We will study frog and mouse NMJs. The frog NMJ is useful because it is one of the best understood synapses, and it is optimally suited for developing and refining the measuring techniques that we will use. The mouse NMJ offers the opportunity to examine genetic knockouts; we will focus in particular on The Synapsin Hypothesis, by comparing vesicle mobilization in synapsin knockout and wild type mice. The techniques that we will use include electrophysiology, electron microscopy, and especially fluorescence microscopy. Both Fluorescence Recovery After Photobleaching (FRAP) and Image Correlation Spectroscopy (ICS) will be employed to quantify vesicle mobilization. Implications for human health: Synapses are like transistors in a computer - the points at which converging information is sorted and analyzed. A synapse is far more complex, however, than a transistor, comprising thousands of different molecules, and even moving parts (the subject of our research). Nearly all therapeutic psychoactive drugs act at synapses. A better understanding of the fundamental mechanisms of vesicle recycling will be useful in understanding diseases of the nervous system, and in developing better drug therapies.